Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2021

Departement Biomedische Wetenschappen

Multiple myeloom bestrijden met eiwitkinase en ferroptose therapieën - Emilie Logie (10/12/2021)

Emilie Logie

  • 10 december 2021, 16u30 - 18u30
  • CDE, Auditorium O1
  • Promotor: Wim Vanden Berghe

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Abstract (Engels)

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of a clone of malignant plasma cells in the bone marrow. Despite recent advances in treatment regimens, MM remains an incurable disease with conventional therapies, such as proteasome inhibitors, immunomodulatory drugs, corticosteroids, and alkylating agents only resulting in low remission rates and limited survival times. This treatment failure of existing anti-cancer drugs can be explained by the development of (acquired) multi-drug resistance, which renders cancer cells cross-resistant to structurally and functionally unrelated drugs. Due to the multifactorial nature of the molecular mechanisms involved in acquisition of therapy resistance, it remains challenging to develop effective, curative treatments for MM. Thus, there is an urgent need to explore novel therapeutic strategies to achieve complete and persistent tumor remission in MM patients. In this PhD thesis work, we explored kinase inhibition, treatment with natural compounds, and ferroptosis induction as alternative therapy strategies to overcome MM drug resistance.

Onderzoek naar de rol van hormonen in de seizoensgebonden neuroplasticiteit in volwassen zangvogels - Jasmien Orije (07/12/2021)

Jasmien Orije

  • 7 december 2021, 17u30 - 19u30
  • CDE, Aula Fernand Nédée (Q.001)
  • Promotoren: Annemie Van der Linden, Marleen Verhoye

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Abstract (Engels)

Early in life neuroplasticity and learning occur more readily than at older age. These so called ‘sensitive periods’ of neuroplasticity have been of particular interest to scientists who tried to change the timing or reintroduce plasticity later in life. Interestingly, seasonal songbirds like starlings, present a seasonal neuroplasticity enabling them to modify their song each year. This makes them a remarkable animal model to study the permissive circumstances for naturally reoccurring neuroplasticity, especially in relation to vocal learning. However, research on neuroplasticity in songbirds mainly focused on the male song control system (SCS), because in many songbird species, song is a predominantly male characteristic.

This PhD thesis aimed (1) to uncover the exact time window when heightened seasonal neuroplasticity emerges in male and female starlings and (2) to investigate the neuromodulatory impact of steroid and thyroid hormones during this time window. Using a non-invasive technique: Diffusion tensor imaging, allowed us to monitor the same animals longitudinally and investigate the microstructural changes within the entire brain in an ‘unbiased’ data-driven way as they experienced different photoperiods and hormone modulations.

This way we discovered that microstructural changes are not limited to the SCS, also other sensory systems and the cerebellum present seasonal neuroplasticity, indicating the importance and integration of multiple sensory systems in preparation of the breeding season. Importantly, this neuroplasticity started during the photosensitive phase, which we therefore define as a sensitive period of multisensory neuroplasticity.

Next, we implanted testosterone in photosensitive female starlings to determine its effects on neuroplasticity and song. We uncovered that testosterone rapidly stimulates the song rate, but other song characteristics, like song bout length, and the microstructural changes in the SCS develop more gradually. Furthermore, this testosterone-induced increase in song bout length correlated with SCS microstructure.

Finally, we examined the role of thyroid hormones on seasonal neuroplasticity. We showed seasonal variations in the expression of several thyroid hormone regulating genes within the SCS, indicating their potential for directly affecting the SCS neuroplasticity. Furthermore, circulating thyroid hormone levels were negatively correlated to microstructural changes in several SCS nuclei, indicating that a reduction in thyroid hormones is necessary to lift the brakes imposed by the photorefractory period and reopen the window of neuroplasticity.

The insights gathered from these experiments tell us something about the permissive circumstances allowing neuroplasticity, setting the stage for further molecular research and encourage to consider the effects of hormones in other forms of neuroplasticity.


De geschiktheid van hSC- en hiPSC-afgeleide hartcellen als in vitro cel model dat de fysiologische omstandigheden voor cardiotoxiciteitsscreening vertegenwoordigt - Dieter Van de Sande (30/11/2021)

Dieter Van de Sande

  • 30 november 2021: 16u - 18u
  • CDE, Promotiezaal (Q.002)
  • Promotoren: Dirk Snyders, Alain Labro

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Abstract (Engels)

Cardiotoxicity is one of the most prevalent unwanted side effects of new drugs in development. To improve the early detection of these side effects, an in vitro model that captures the in vivo physiology is desired in the non-clinical safety screening of new drug compounds. A promising model are human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). These hiPSC-CMs express a broader repertoire of cardiac proteins than expected for heterologous cell expression systems, which overexpress one protein of interest. Consequently, a more physiological model is created. However, the model is still too immature compared to native CM as their morphology and electrophysiological parameters correspond more to an embryonic like state. In this thesis the usability of the model for cardiotoxicity screening is further researched with the main focus on electrophysiology. The effect of the β1 subunit on the cardiac sodium current and its pharmacological profile was determined. As the hSC-CM model expresses the beta subunit it could indicate if the model is more suitable to detect Nav1.5 channel pharmacology compared to the heterologous expression models. While five compounds were tested only lidocaine and phenytoin had reduced IC50 when the β1 subunit was present in the heterologous model. Interestingly the IC50 of the hSC-CM model was comparable which indicates the functional expression of the subunit and the ability of the model to accurately detect Nav1.5 pharmacology. At the same time it was also observed that the RMP value of the action potential waveform determined the availability of the Nav1.5 channels for activation. RMP potentials of -50 mV in isolated hSC-CMs did not result in a sufficient upstroke velocity. However, when patching hSC-CM in a syncytium the RMP value is more hyperpolarised compared to isolated hSC-CMs. This resulted in a more physiological upstroke velocity, indicating the necessity of cell to cell contacts to create a more physiologically relevant model. In a last part the effect of membrane cholesterol on the pharmacological profile is determined to research the possible need of a hypercholesterolemia model in cardiotoxicity screening. However, no significant differences were observed for the drug quinidine and further experiments are needed to create a solid statement. In general the hSC-CM is a strong model for cardiotoxicity screening when working with syncytium’s and can detect most possibly the effect of auxiliary subunits on the pharmacological profile of ion channels.  Which will also reduce the use of laboratory animals in preclinical screening.

Gedetailleerde analyse van nanobubbel gemedieerde disruptie ter hoogte van de plasma membraan en kernenvelop - Gaëlle Houthaeve (16/11/2021)

Gaëlle Houthaeve

  • 16 november 2021, 17u30 - 19u30
  • CDE, Promotiezaal (Q.002)
  • Promotoren: Winnok De Vos, Kevin Braeckmans

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Abstract (Engels)

Vapor nanobubble (VNB)-photoporation allows to deliver various compounds into a broad range of cell types with minimal toxicity. It relies on laser irradiation of plasma membrane (PM)-adhered gold nanoparticles (AuNPs), which results in the formation of VNBs. The subsequent collapse of VNBs near the PM facilitates its transient disruption, whereby cargo can enter the cytosol. Intracellular delivery is desirable in various biomedical applications, such as the delivery of nucleic acids for genetic engineering of cells for therapy. Despite its promise, however, to date little is known about the effects of VNB photoporation on cell physiology. Elucidation of the cellular response to VNB-induced PM disruption could aid in the development of strategies to diminish side effects and thereby increase its efficiency.

To uncover the cellular response to VNB photoporation, we performed a longitudinal RNA sequencing experiment. We found a marked upregulation of the LMNA gene 6h after photoporation, which had disappeared at later timepoints. This was mirrored at the protein level (A-type lamins), and was accompanied by a temporary increase in chromatin condensation. We found this nuclear stiffening to be required for the cell to cope with the effects of VNB photoporation. Correspondingly, experimental increase of A-type lamin levels was able to increase the fraction of successfully transfected cells.

The versatile nature of VNB photoporation motivated us to explore its potential for use at the nuclear envelope (NE). Transient ruptures of the NE (NERs) have recently been found to occur in laminopathy patient cells and cancer cells. Although cells are able to repair these NERs, the uncontrolled exchange of nuclear and cytoplasmic factors will undoubtedly have consequences on cell physiology. However, detailed investigation of the effects of NERs is currently hampered by their stochastic nature and variable frequency. Therefore, we set out to adapt VNB photoporation for the transient disruption of the NE.

We demonstrated that perinuclear localization of AuNPs can be achieved after endocytic uptake or electroporation-facilitated delivery, and that both strategies result in NER upon laser irradiation. We found that the induced NERs mimicked spontaneous NERs to a high degree. We also demonstrated that NE photoporation is able to allow the intranuclear delivery of compounds that are otherwise not able to cross the NE. Once fully optimized, NE photoporation will not only be a substantial asset for research on NERs, it will become a valuable tool for a wide range of applications that require the transient and controlled disruption of the NE.

Farmacologie van nieuwe geneesmiddelen tegen viscerale leishmaniasis en Afrikaanse slaapziekte - Magali Van den Kerkhof (30/09/2021)

Magali Van den Kerkhof

  • 30 september 2021, 16u - 18u 
  • CDE, Promotiezaal (Q.002)  
  • Promotoren: Guy Caljon, Louis Maes

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Abstract

Viscerale leishmaniasis (VL) en slaapziekte (HAT: human African trypanosomiasis) zijn verwaarloosde, tropische ziektes die veroorzaakt worden door respectievelijk Leishmania infantum of L. donovani en Trypanososma brucei gambiense of T. b. rhodesiense. Beide parasieten worden overgedragen door bloedzuigende insecten en veroorzaken systemische infecties die kunnen leiden tot hoge morbiditeit; zonder behandeling leiden deze infecties overigens tot sterfte. De huidige behandelingen voor VL en HAT hebben een eerder lage werkzaamheid, vertonen ernstige neveneffecten, moeten parentaal toegediend worden en ondervinden steeds meer last van therapiefalen. Nieuwe geneesmiddelen zijn noodzakelijk om patiënten op een meer adequate manier te behandelen en om het succes op uitroeiing van deze ziektes te vergroten. Recente initiatieven hebben een aantal veelbelovende nieuwe teststoffen geïdentificeerd tegen leishmaniasis, zoals de nitroimidazoles, aminopyrazoles en oxaboroles, alsook tegen slaapziekte, zoals de pyrazolopyrimidinone phosphodiesterase inhibitoren.

Een eerste doel van dit proefschrift was het evalueren van de farmacodynamiek van deze nieuwe teststoffen. De anti-Leishmania stoffen vertoonden een potente en selectieve activiteit, zowel in vitro als in vivo zonder kruisresistentie met de huidig gebruikte medicijnen. De uitgesproken in vitro activiteit van de pyrazolopyrimidinones kon helaas niet bevestigd worden in muis modellen: parasieten werden niet geëlimineerd uit alle organen en toxiciteit was een probleem in het chronisch model.

Het tweede doel was om het werkingsmechanisme (WM) en resistentiemechanisme (RM) van deze teststoffen te bepalen. Resistentieselectie gevolgd door sequenering, cosmid-sequenering, metabolomics,  en een recentere proteomics techniek, genaamd DARTS, werden gebruikt om het WM/RM van de nieuwe teststoffen te onderzoeken. Deze experimenten hebben helaas niet geleid tot het identificeren van exacte doeleiwitten, maar hebben meerdere indicaties gegeven van mogelijks interessante genen. Het WM van de pyrazolopyrimidinones werd onderzocht met een genoom-brede “knock-down” parasieten collectie en een metabolomics experiment. Beide technieken toonden aan dat de S-adenosylmethionine (Adomet) pathway beïnvloed werd, maar opvolgexperimenten konden dit niet bevestigen.

Besluit: het is duidelijk dat de nitroimidazoles, aminopyrazoles en oxaboroles veelbelovende anti-Leishmania ontwikkelingskandidaten zijn die wellicht geen of weinig problemen zullen ondervinden met resistentie ontwikkeling. Bovendien kunnen deze in combinatietherapieën ingezet worden wegens hun verschillend werkingsmechanisme ten opzichte van elkaar en de huidige geneesmiddelen. Hun exacte WM/RM moet nog verder worden onderzocht aan de hand van complementaire technieken. De pyrazolopyrimidinones daarentegen vereisen verdere optimalisatie om een betere en meer veilige werkzaamheid te bekomen in zowel het acuut als het chronisch stadium van HAT. De identificatie van de Adomet pathway kan inspireren in de zoektocht naar nieuwe, meer potente alternatieven met verminderde toxiciteit.

Moleculair en functioneel studie van twee frequente syndromale Autisme Spectrum Stoornissen: Helsmoortel-Van der Aa Syndroom and Fragile X Syndroom

Elisa Cappuyns

  • 21 september 2021, 17u - 19u
  • CDE, Auditorium O3 (na registratie via student) 
  • Promotoren: Frank Kooy, Geert Vandeweyer

Toegang is enkel mogelijk na voorafgaande kennisgeving van uw naam en emailadres bij de student EN op vertoon van uw ID en geldig Covid Safe Ticket vóór het binnengaan van het lokaal.  Registratie start om 16u30 en is slechts mogelijk tot aan de start van de verdediging! 

Abstract (Engels)

Neurodevelopmental disorders enclose a heterogeneous group of conditions implicating central nervous system development. The majority of cases are caused by genetic defects. Mutations in the genes encoding Activity-Dependent Neuroprotective Protein (ADNP) and Fragile X Mental Retardation Protein (FMRP) make up the two common genetic causes of ASD combined with ID. Although both are ASD/ID syndromes, their genetic mechanism is completely different and the focus of this thesis.

In a first part of this thesis, we elaborate on the impact of ADNP mutations. A first objective was finding a reliable ADNP detection method to evaluate its stability and functioning upon mutation. We tested several ADNP antibodies. The blocking peptide strategy enabled us to differentiate between specific and non-specific signals in different specimens. With a GFP-ADNP expression vector, we detected a shorter, mutant ADNP protein. Our results verify the presence of mutant forms of ADNP, caused by Helsmoortel-Van der Aa-like mutations.

In a second objective, we studied correlations between the distinct ADNP mutations and their impact on the protein, by examining the expression and subcellular localization of GFP-tagged mutant transcripts in transfected HEK293T cells. We found mutations presenting a pattern based on the genetic position. Mutations affecting the nuclear localization signal caused mislocalization of mutant protein in the cytoplasm. N-terminal truncated ADNP mutants are routed towards cytosolic proteasomal degradation and rescued with the proteasome inhibitor MG132. C-terminal mutants presented wildtype-like nuclear localization, but those with an arrested HP1α interaction motif showed decreased affinity for heterochromatin interaction. Our results suggest a correlation between the position of the mutations, protein stability and subcellular localization.

The third objective was to generate and validate a “humanized” (carrying a human mutation) mouse model to further study HVDAS-related mutations on a functional level. Using Crispr/Cas9, we introduced a frameshift mutation near a clustering site of known patient mutations. We assigned the mutant mouse model to a standardized behavioral test battery. Our model presented similarities to HVDAS patients, including impaired cognitive functioning, increased anxiety and repetitive behavior.

The second part of this thesis focuses on kinome analysis in Fmr1 knockout mice, a model for Fragile X Syndrome (FXS), using the PamGene platform. We observed a general upregulation of kinase activity in an Fmr1 knockout mice. Through upstream kinase analysis, we observed dysregulation in glycogen and immune pathways. Furthermore, we found statistical evidence for the dysregulation of 46 proteins. These candidates have to be validated for biomarker potency for future drug testing.

Het verbeteren van de diagnose en controle van seksueel overdraagbare aandoeningen bij mannen die seks hebben met mannen in België - Irith De Baetselier (13/09/2021)

Irith De Baetselier

Abstract

Het aantal bacteriële seksueel overdraagbare aandoeningen (soa's) onder mannen die seks hebben met mannen (MSM) neemt de laatste tien jaar dramatisch toe. De toename van soa's (gonorroe, chlamydia of syfilis) kan vooral versterkt worden door PrEP-gebruikers die herhaaldelijk met soa's besmet raken (recidiverende soa's). PrEP of pre-exposure prophylaxis is een biomedische preventiemethode om hiv te voorkomen en kan leiden tot risicovoller seksueel gedrag, zoals een toename van condoomloze anale seks, meer seksuele mixing tussen hiv-negatieve en -positieve MSM en/of een toename van gelijktijdige seksuele partners. Personen met recidiverende soa's kunnen cruciale posities innemen in omvangrijke seksuele netwerken. Daarom kan het identificeren van deze individuen van belang zijn voor de volksgezondheid om de keten van transmissie te doorbreken. In deze thesis hebben we de soa-epidemie onder Belgische MSM PrEP-gebruikers onder de loep genomen en hebben we de gedragsfactoren geïdentificeerd die geassocieerd zijn met terugkerende soa's, zoals geseksualiseerd druggebruik: dit werd gerapporteerd bij bijna 90% van de individuen die herhaaldelijk besmet waren met soa’s. Bovendien bleven bijna alle Lymphogranuloma venereum (LGV) infecties, een opkomende soa, beperkt tot de groep met recidiverende soa's.

Momenteel bevelen de Belgische PrEP-richtlijnen driemaandelijkse soa screening aan bij MSM. Door de huidige budgettaire beperkingen en de zware belasting van frequente staalafnames voor gezondheidswerkers, laboratoriummedewerkers en PrEP-gebruikers, onderzochten we nieuwe, kosteneffectieve strategieën voor soa-screening, zoals zelfafnames thuis en poolingstrategieën. Ten slotte werd een nieuwe soa-screeningstrategie ontwikkeld gericht op individuen die herhaaldelijk geïnfecteerd zijn met soa’s in plaats van op alle PrEP-gebruikers.

Naast de toename van soa's, kunnen Neisseria gonorrhoeae en Mycoplasma genitalium (MG) zich ontwikkelen tot "superbugs". In dit proefschrift onderzochten we de prevalentie van resistentie tegen de eerste- en tweedelijnsbehandeling van MG onder PrEP-gebruikers en vonden we een opmerkelijk hoge prevalentie van resistentie tegen macroliden (88%) en fluorochinolonen (26%). Een extra analyse toonde aan dat deze hoge aantallen resistentie veroorzaakt werden door de MG-gevallen die geïdentificeerd werden bij MSM met recidiverende soa's.

Concluderend kan worden gesteld dat dit proefschrift de centrale rol blootlegt die personen met recidiverende soa's hebben in de soa-epidemie onder MSM. Daarom wordt frequent soa-onderzoek, behalve op MG, nog steeds aanbevolen, maar dan toegespitst op deze groep. De nieuwe algoritmen voor soa-testen die in dit proefschrift zijn ontwikkeld, zullen de last van het frequent testen op soa's onder PrEP-gebruikers helpen verminderen zonder de kwaliteit van zorg te verlagen.

Opheldering van de rol van GSDME tijdens secundaire necrose na apoptose - Elke De Schutter (8/9/2021)

Elke De Schutter

  • 8 september 2021, 16u - 18u
  • CDE, Auditorium O5 (na registratie via student) + ONLINE
  • Promotoren: Guy Van Camp, Peter Vandenabeele, Frank Riquet

Abstract (Engels)

Although clear evidence exists for the contribution of gasdermin E (GSDME) to hearing loss and tumor biology, the physiological function of GSDME and how it could explain a role in both disorders remained unclear. As GSDME was reported to be a substrate of the apoptotic caspase-3 and is proposed to act as a pore-forming molecule, we aimed in this thesis to unravel the role of GSDME in apoptosis-driven secondary necrosis. Therefore we explored different aspects both in silico and in vitro. Using homology-based modeling, we investigated the composition and functionality of secondary structures in the GSDME protein and compared our model to the published structures of other gasdermin (GSDM) proteins. Next, we measured the GSDME-dependent nuclear staining by DNA dyes and the influx of Texas Red-labeled dextrans during apoptosis-driven secondary necrosis in order to gain more insight about the kinetics and size characteristics of GSDME-dependent cell death. Finally, we monitored the cellular localization of GSDME during apoptosis-driven secondary necrosis using live-cell imaging. Our results show that GSDME facilitates the quick permeabilization of the plasma membrane after activation of caspase-3 favoring the staining by SYTOX dyes and Texas Red-labeled dextrans. However, we also found that GSDME is probably not the only executor of membrane permeabilization acting downstream of caspase-3 activation, as the nuclear staining by 7-AAD and efflux of FITC-labeled dextrans happened independent of GSDME expression during apoptosis-driven secondary necrosis. Future research should focus on the consequences of GSDME-mediated plasma membrane permeabilization in terms of release of pro-inflammatory molecules and clearance by phagocytic cells. In addition, our results question the universality of the barrel-stave pore model as the pore-forming mechanism of GSDM proteins. Further comparative analysis with other known pore-forming molecules should be done to provide more certainty about the pore-forming mechanism of GSDME.



Systematische beeld-gebaseerde profilering van neuronale connectiviteit. Van de synaps tot de celkern. - Marlies Verschuuren (28/06/2021)

Marlies Verschuuren

  • 28 juni 2021, 16u30 - 18u30
  • Online verdediging
  • Promotor: Winnok De Vos

Abstract

Long-term adaptations of the brain, including learning, memory formation, addiction and chronic pain sensation, rely on a delicate balance of tunable connections between neurons. In numerous neurological diseases this neuronal connectivity is perturbed. Neuronal connectivity is classically assessed by means of morphological correlates such as the density of synapses or functional readouts such as synchronous calcium spiking activity. However, such approaches operate with blinders as they do not account for multifaceted effects. Furthermore, recent reports have inferred an important role for the nucleus in neuronal connectivity. As yet, nuclear morphology has not been regarded as a potential readout of neuronal connectivity and limited tools are available to capture it with sufficient sensitivity.

We established a detailed interrogation of morphological and functional correlates of neuronal connectivity in one single microscopy-based assay to screen for its modulators in primary neuronal cultures. The integrative approach proved to be more sensitive than more classical single-readout approaches. Using this pipeline, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures, albeit within a specific time and dose range. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed, suggesting it has broad-spectrum therapeutic relevance. In addition to this integrated assessment of neuronal connectivity, we optimized an image analysis algorithm that accurately detects nuclei in fluorescence images and captures their morphology by precise delineation of their contour and by extraction of robust, quantitative descriptors.

Having established a method to interrogate nuclear morphology and to connect it with neuronal connectivity, we next sought to evaluate the influence of targeted perturbations to nuclear morphology. Given their crucial role in nuclear architecture and function, we thereby focused on the nuclear lamins. A significant decrease in lamin B1 levels with culture time and brain aging, drove us to interrogate the effect of its selective depletion in human neuroblastoma model cells using shotgun proteomics. In-depth analysis revealed that lamin B1 acts as a molecular switch controlling differentiation, by tempering integrated stress levels.

The results that have been generated in this thesis, illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. Our work also proves that nuclear lamins have a crucial role in steering neuronal cell function and may be considered as a putative target for novel modulators of neuronal connectivity.

Biomerkers van maladaptieve plasticiteit tijdens epileptogenese met in vivo hersenbeeldvorming - Daniele Bertoglio (28/06/2021)

Daniele Bertoglio

  • 28 juni 2021, 18u - 20u
  • Online verdediging 
  • Promotoren: Annemie Van der Linden, Jeroen Verhaeghe, Stefanie Dedeurwaerdere

Abstract

​Epilepsy is one of the most common chronic neurological conditions affecting more than 65 million people worldwide. It may be genetic or acquired following different types of brain injury, such as status epilepticus (SE), trauma, or stroke. Temporal lobe epilepsy (TLE) represents the most common form of drug-resistant focal epilepsy in adults. In TLE, the initial brain insult is followed by a latent period during which several pathophysiological changes occur, including brain inflammation, impairment of networks, and synaptic circuit alterations. Ultimately, these modifications result in the transition of a healthy brain into a hyperexcitable one leading to the development of spontaneous recurrent seizures. This process is defined as epileptogenesis. 

The seizure-free period lasting between the epileptogenic insult and the clinical onset represents a therapeutic window of opportunity for disease-modifying therapies aimed at halting or preventing the process of epileptogenesis. Despite the attractive therapeutic paradigm, the development of (pre)clinical studies to investigate the efficacy of antiepileptogenic treatments is severely hampered by the wide heterogeneity in insults and our inability to predict and stratify subjects at risk. 

This thesis aims to characterize novel clinically relevant in vivo imaging markers of epileptogenesis in the kainic acid-induced status epilepticus (KASE) rat model of TLE to identify prognostic markers for the long-term disease outcome. Specifically, using positron emission tomography (PET) and magnetic resonance imaging (MRI), we focused on the assessment of translocator protein 18kDa (TSPO) upregulation as a proxy for neuroinflammation as well as functional connectivity changes as a read-out for the epileptogenic neuroplasticity. 

First, we compared the KASE rat model of TLE to select the most appropriate strain to elucidate pathological changes during epileptogenesis given the divergences in disease progression and seizure outcome. Secondly, we demonstrated that a single TSPO PET scan at disease onset (2 weeks post-SE) could accurately predict the seizure frequency of each epileptic rat, suggesting its potential prognostic value for the stratification of subjects at risk, differentiating categories of epileptic rats with significantly distinct seizure burden. Lastly, by investigating functional network connectivity changes during epileptogenesis, we observed a widespread network hyposynchrony at disease onset as well as diverging changes depending on disease severity. 

In conclusion, the work of this thesis identified relevant preclinical prognostic imaging markers of epileptogenesis predictive of seizure frequency. Future studies will be necessary to understand whether these markers might be utilized clinically.

Verbetering van de diagnose van rifampicine-resistente tuberculose: programmatische aspecten, diagnostische uitdagingen, en moleculaire epidemiologie - Semuto Jean Claude Ngabonziza (23/06/2021)

Semuto Jean Claude Ngabonziza

  • 23 juni 2021, 16u - 18u
  • Online verdediging: https://Itg.zoom.us/j/85897541694?pwd=K0RtUlh2aGlQOVhLQnlKazAxaWxuUT09
  • Promotoren: Leen Rigouts, Bouke de Jong
  • Abstract

    Tuberculosis (TB) remains the leading infectious disease cause of mortality globally. Resistance to rifampicin (i.e. the most powerful anti-TB drug) impedes optimal management of patients and TB control. Indeed, the majority of rifampicin-resistant (RR) TB patients remain undiagnosed and untreated, thus spreading resistant Mycobacterium tuberculosis complex (MTB) strains. In Rwanda  the first RR-TB patients were diagnosed in 1989. However,  adequate programmatic management of RR-TB started 15 years later, in response to an increasing prevalence and poor treatment outcomes. Stepwise interventions were implemented aiming at early diagnosis and appropriate treatment. First, the program expanded access to culture-based RR-TB detection. Second, rapid molecular tests, such as Xpert MTB/RIF, were implemented to swiftly diagnose RR-TB. My Ph.D. research showed a substantial reduction of delays in initiating RR-TB treatment from 175 days in 2006 to 5 days only in 2016. This reduction of delays decreased RR-TB related mortality from 30.8% in 2006 to 6.9% in 2016. Besides reduced mortality, I showed that such swift detection interrupted the spread of RR-TB, also that most RR-TB in Rwanda was caused by a single MTB clone that we named ‘Rwanda rifampicin-resistant TB clone’, or ‘R3clone’. Through universal access to rapid rifampicin resistance testing, the R3clone population declined since 2014. However, the extensive use of Xpert MTB/RIF did not only yield positive effects. Ten years after its global rollout, my analysis showed very important pitfalls of Xpert MTB/RIF. As more and more patients were tested with Xpert MTB/RIF, a substantial proportion of patients were diagnosed early with paucibacillary disease. I showed that half of these patients diagnosed with RR-TB in fact had rifampicin-susceptible TB. The Xpert MTB/RIF software erroneously interprets insufficient DNA binding as evidence of resistance. Unfortunately, these patients were unnecessarily treated with a longer ‘second line’ treatment regimen with more toxic drugs. Based on my findings the National TB Programme in Rwanda changed the diagnostic algorithm, to further ascertain RR in patients with a paucibacillary sample before starting RR-TB treatment. Today, TB patients in Rwanda only receive second-line treatment if they really need it. Besides the challenges in diagnosing RR-TB, I identified a novel MTB lineage that we named lineage 8 (L8). The L8 is a sister clade to the known MTB lineages. Remarkably, the two L8 strains identified so far were resistant to key anti-TB drugs. L8 seems to be extremely rare and restricted to the Great Lakes region.

Ontwikkeling van efficiënte therapieën voor vasculaire media calcificaties, een belangrijk klinisch probleem in onze vergrijzende maatschappij - Britt Opdebeeck (31/05/2021)

Britt Opdebeeck

  • 31 mei 2021, 16u30 - 18u30
  • Promotiezaal (CDE) of ONLINE
  • Promotoren: Patrick D'Haese, Anja Verhulst

Abstract

Arterial media calcification or the deposition of calcium-phosphate crystals (i.e. hydroxyapatite) in the medial layer of the arterial wall is a major cardiovascular complication in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. This pathology favors arterial stiffness, hypertension and left ventricular hypertrophy ultimately leading to heart failure, impaired coronary perfusion and cardiac stroke. Moreover, CKD patients with known arterial calcification have to be considered at highest cardiovascular risk. As no effective treatment strategy is available at this time, finding novel and efficient therapies that directly target the mineralization process in the vessel wall are urgently needed. The first research objective focused on the role of protein-bound uremic toxins in the arterial media calcification process. This objective revealed that, in a context of CKD, protein-bound uremic toxins, indoxyl sulfate and p-cresyl sulfate, directly promoted arterial media calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis. The second research objective investigated the role of extracellular nucleotides in arterial media calcification through activation of purinergic independent (tissue-nonspecific alkaline phosphatase (TNAP) inhibition) and dependent (P2X1 receptor) pathways. The enzyme TNAP mediates the hydrolysis of calcification inhibitor pyrophosphate (a metabolic degradation product of nucleotides) into the calcification stimulator inorganic phosphate. We found that a dosage of 10 mg/kg/day TNAP-inhibitor SBI-425 was sufficient to inhibit the development of mild arterial media calcification (i.e. warfarin rat model) however, turned out to be insufficient for treating moderate to severe CKD-related arterial media calcification (i.e. adenine rat model). Also treatment with TNAP-inhibitor SBI-425 resulted into bone mineralization side-effects. Similarly, P2X1 receptor agonist b,y-meATP prevented the development of mild arterial media calcification (i.e. warfarin rat model) however provoked also deleterious effects on bone mineralization. Since patient populations developing arterial media calcification (CKD, diabetes as well as osteoporosis patients) already suffer from a seriously compromised bone health this is of significant clinical importance. Therefore, exploring new therapeutic targets related to inflammation, coagulation and/or glucose homeostasis, investigating the dissimilarities between the calcification process in the arteries and bone, as well as trying to selectively target compounds such as β,γ-meATP to the blood vessels by using i.e. nanoparticle conjugated to antibody drug delivery system might all be promising ways to obtain safe and efficient anti-arterial calcification therapies.

Een proteoom gebaseerde beschrijving van de Withaferin A antitumor eigenschappen - Martin Dom (12/03/2021)

Martin Dom

  • 12 maart 2021, 16u - 18u
  • Online verdediging
  • Promotoren: Xaveer Van Ostade, Wim Vanden Berghe

Abstract

Ondanks de veelbelovende multifunctionele antitumor eigenschappen van Withaferine A (WA), ontbreekt een algemeen overzicht van de onderliggende moleculaire antitumor mechanismen. Dit zet een rem op het potentieel klinisch gebruik van WA. Multifunctionele chemotherapie is een veelbelovende strategie in het bestrijden van multidrug resistente tumoren en we wilden met deze thesis de kennis leegte tussen WA behandeling en de geobserveerde kanker celdood opvullen.

Om deze leemte in kennis op te vullen, hebben we een kwantitatieve chemoproteomics workflow ontwikkeld waarmee we de eiwit bindingspartners van WA hebben gekarakteriseerd. Deze data, gecombineerd met differentiële eiwit expressie data, hebben ons tot de hypothese geleid dat WA een acute stress induceerder is én tegelijk de stress homeostasis response als doelwit heeft. Hiertoe behoren o.a. de oxidatieve stress response, het ubiquitine-proteasome systeem, autofagie en ERAD response. Zelfs wanneer kanker cellen de basale cellulaire stress levels herstellen, dan beïnvloedt WA ook meer structurele, non-stress, cellulaire events. Hiertoe behoren o.a. cytoskelet verzwakking, onderbreken van de cel cyclus en van de Akt/NFkB pathways. Verder identificeerden we een nieuwe manier van WA proteasome inhibitie die verloopt via de binding met de deubiquitinerende enzymes USP14 en UCHL5.

We geloven dat onze resultaten aantonen dat de technologische vooruitgang ons toelaat om target-based en observatiegebaseerde drug ontwikkeling strategiën te combineren en zo een heel nieuw veld van antitumorale therapiën te verkennen.

Strategieën voor het efficiënt opnemen en reconstrueren van structurele en quantitatieve MR beelden - Maarten Naeyaert (11/02/2021)

Maarten Naeyaert

  • 11 februari 2021, 17u - 19u
  • Online verdediging
  • Promotoren: Marleen Verhoye, Jan Sijbers

Abstract

Magnetic resonance imaging (MRI) is a valuable tool for investigating the brain, both in the clinic and in research. Its main drawback is its long acquisition time, which can be shortened by minimising the amount of data which is acquired and by using it efficiently. Three objectives were set for this thesis, each of which makes efficient use of the acquired data.

Two objectives relate to compressed sensing (CS). CS algorithms allow the reconstruction of signals or images from incomplete data, by acquiring this data in a pseudo-random way and finding the most compressible image still consistent with the acquired data. The final image quality depends on which data is sampled, and several acquisition strategies were tested on both simulated signals, test objects, and using actual acquisitions. Firstly, CS was implemented for a fast spin echo (FSE) sequence, and it was found that optimal results occur when the majority of the data is sampled in the centre of k-space, with progressively less data being sampled closer to the edge of k-space.

Secondly, a method to accelerate the acquisition of isotropic in-vivo high-angular radial diffusion imaging (HARDI) data using an FSE sequence was developed. While slow, the FSE sequence results in few artefacts, and CS can be used to acquire the large number (>50) of volumes required for HARDI faster. In this case, CS can also be used to subsample the diffusion signal, in the q-space, to acquire fewer volumes. Using knowledge from the first objective, strategies for subsampling the data, either only in q-space or both in k-space and q-space, were tested. We found that subsampling the q-space only is most efficient, and 15 to 20 volumes proved sufficient to yield high quality reconstructions without major differences in quantitative diffusion measures or tractography results.

Finally, a method was developed to quantify micron-sized iron-oxide particles (MPIO) in MRI-images. These particles are used to label cells and cause a negative contrast, i.e. hypo-intensities. However, positive contrast images can be constructed to improve the visibility and localisation of MPIO, which was done efficiently from a single acquisition. To quantify MPIO robustly and reliably the normalized average range (nAR) is introduced. The nAR compares the average value of regions of interest (ROI’s) to that of a control ROI in range filtered images, and showed a higher sensitivity in optimized positive contrast images and is not sensitive to the bias field of the receiver coil.

Verdere inzichten in de pathogenetische mechanismen van melorheostose en de ziekte van Paget - Raphaël De Ridder (26/01/2021)

Raphaël De Ridder

  • 26 januari 2021, 16u30 - 18u30
  • Online verdediging
  • Promotoren: Wim Van Hul, Geert Mortier

Abstract

Dysregulation of bone remodeling can lead to a large number of skeletal disorders. Elucidating the molecular background of such pathologies has the potential of improving diagnosis and can be of great value in the identification of novel targets for the development of novel therapies. The general aim of this thesis was therefore to investigate the contribution of genetic factors in the pathogenesis of two bone disorders, melorheostosis and Paget’s disease of bone (PDB).

In a first part, we performed a mutation analysis in a small cohort of melorheostosis cases. This is a very rare condition characterized by asymmetric increased density of the cortical bone and alteration in surrounding soft tissues. This analysis confirmed the importance of somatic pathogenic variation in the negative regulatory region of MAP2K1 and showed the presence of a novel melorheostosis-associated somatic p.Cys121Ser variant in the catalytic core. Gene-set enrichment of transcriptomic data indicates that this variant leads to hyperactivation of proliferative pERK signaling. 

The second part of this thesis was focused on PDB. Follow-up of families with a history of PDB further supports the importance of genetic variation in SQSTM1. Based on the molecular background of a number of PDB-related phenotypes, targeted sequencing was performed for a panel of 52 candidate genes. This indicated a significant association for variation in the RIN3 gene with the phenotype, with the rs117068593 variant conferring a protective effect that modifies the age of onset in patients. A panel-wide gene burden analysis highlighted association for a number of genes (TNFRSF11A, NUP205, VCP and NFKBIA) with PDB pathogenesis and suggested an enrichment of rare genetic variation in several other genes (NR4A1, NUP205 and PRKCI). Finally, the use of next-generation sequencing technologies is rapidly expanding the number of candidate genes that requires further functional evaluation. Therefore, we generated a loss-of-function model of the ubiquitin-associated domain in the sqstm1 gene of zebrafish. Preliminary results support the presence of a skeletal phenotype in our model. Further studies will be needed to elucidate whether this reflects an osteoclast-driven Pagetic phenotype.

Together, our results provide further insights into the regulation of bone remodeling and explore a novel platform to study the pathogenesis of skeletal disease in vivo.

Drug-resistentie tijdens viscerale leishmaniasis: impact op de infectiviteit van de parasiet in de gewervelde gastheer en zandvlieg vector - Dimitri Bulté (26/01/2021)

Dimitri Bulté

  • 26 januari 2021, 16u - 18u
  • Online verdediging
  • Promotoren: Guy Caljon, Louis Maes

Abstract

Visceral leishmaniasis (VL) is causing 50,000 to 90,000 new cases annually and more than 20,000 deaths which makes it the deadliest parasitic disease after malaria. In the absence of a successful vaccine, the main VL control strategy is dependent on chemotherapy, however, only a few antileishmanial treatment options are available. Miltefosine (MIL) is currently the only oral drug for VL but is failing to fully clear parasites in an increasing number of patients. These treatment failures could initially not be linked to the emergence of resistance, although more recently a few MIL-resistant (MIL-R) clinical isolates have been described. In order to safeguard the use of MIL for future VL therapies, it is essential to evaluate the impact of MIL-resistance on parasite fitness in the vertebrate host and sand fly vector as this could indicate the potential spread of resistant parasites into the population.

In accordance with previous studies using a laboratory MIL-R L.infantum, a decreased infectivity of the vertebrate host was observed for a natural MIL-R L. infantum. However, no impact on the development of the parasite in the sand fly vector was observed. These results indicate that the impact of resistance on parasite fitness in the vertebrate host and insect vector may not necessarily be the same and indicate the potential transmission of MIL-R parasites. To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo infectivity and drug efficacy, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of laboratory MIL-R and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a relatively lower burden of MIL-R parasites in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites which contributed to the attenuated MIL-R phenotype. In addition, it was demonstrated that the presence of MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. These results highlight the potential risk of MIL treatment in sustaining infections with resistant parasites. Close monitoring of parasite drug susceptibility and adjusted treatment protocols would therefore be beneficial.